Oligomeric compounds having the ability to specifically bind natural and synthetic polynucleotides have numerous uses in analytical methods for detection, identification, and quantification of polynucleotides, as primers and probes for amplifying genes and gene products (e.g. the polymerase chain reaction, PCR), in target validation studies and as therapeutics. Oligomeric compounds such as oligonucleotide DNA and RNA have been used successfully to detect natural polynucleotides and polynucleotide products on so-called biochips. Oligomeric compounds can also be used as primers and probes for taq-polymerase in PCR. Various oligonucleotide compounds and derivatives thereof have been successfully employed in gene-silencing, both in vitro and in vivo. Such oligonucleotide compounds and their derivatives include so-called antisense compounds—oligomers capable of specifically binding a gene or gene product, and either directly or indirectly effecting silencing of the gene.
Antisense therapeutics have shown great promise. Antisense therapeutics modulate protein activities by attenuating the concentration of oligonucleotides, especially RNA, involved in protein synthesis. This is in contrast to conventional therapeutic methods, which seek to modulate protein activities by direct interaction between putative drugs and proteins.
In general, antisense methods involve determining the sequence of a coding oligonucleotide (e.g. mRNA) that encodes for a certain protein (sense strand), developing a relatively short oligomer that selectively binds to the sense strand, and introducing the oligomer into the intracellular environment. Antisense methods can predictably silence gene expression through a variety of mechanisms. In one such mechanism, Translation Arrest, the antisense strand blocks translation by competitively binding to the sense strand of mRNA. In another mechanism, an antisense strand containing a stretch of DNA (e.g. phosphorothioate DNA) binds to the sense strand, whereby the DNA-RNA hybrid is recognized by RNAse H, an endonuclease that selectively cleaves the DNA-RNA hybrid, thereby reducing intracellular RNA levels. Another methodology involves the interaction between small double stranded RNA oligomers and mRNA. In such mechanisms, interaction between the RISC complex, the antisense strand of the small double stranded RNA and intracellular mRNA results in cleavage and degradation of the mRNA.
As antisense molecules have become accepted as therapeutic and diagnostic agents, the need to produce oligonucleotides in large quantities, at higher purity, and at decreased per unit cost has increased as well. The most commonly used antisense compounds to date have been phosphodiester oligonucleotides, phosphorothioate oligonucleotides and second generation oligonucleotides having one or more modified ribosyl sugar units, and more recently, ribosyl sugar units. The methods for making these three types of antisense oligomers are roughly similar, and include the phosphotriester method, as described by Reese, Tetrahedron 1978, 34, 3143; the phosphoramidite method, as described by Beaucage, in Methods in Molecular Biology: Protocols for Oligonucleotides and Analogs; Agrawal, ed.; Humana Press: Totowa, 1993, Vol. 20, 33-61; and the H-phosphonate method, as described by Froehler in Methods in Molecular Biology: Protocols for Oligonucleotides and Analogs; Agrawal, ed.; Humana Press: Totowa, 1993, Vol. 20, 63-80. Of these three methods, the phosphoramidite method has become a de facto standard in the industry.
A typical oligonucleotide synthesis using phosphoramidite chemistry (i.e. the amidite methodology) is set forth below. First, a primer support is provided in a standard synthesizer column. The primer support is typically a solid support (supt) having a linker (link) covalently bonded thereto. It is common to purchase the primer support with a first 5′-protected nucleoside bonded thereto.

Primer support: bg is a 5′-blocking group, Bx is a nucleobase, R2 is H, OH, OH protected with a removable protecting group, or a 2′-substituent, such as 2′-deoxy-2′-methoxyethoxy (2′-O-MOE), and link is the covalent linking group, which joins the nucleoside to the support, supt.                (A) The 5′-blocking group bg (e.g. 4,4′-dimethoxytrityl) is first removed (e.g. by exposing the 5′-blocked primer-support bound nucleoside to an acid), thereby producing a support-bound nucleoside of the formula:        
Activated primer support: wherein supt is the solid support, link is the linking group, Bx is a nucleobase, R2′, is H, OH, OH protected with a removable protecting group, or a 2′-substituent.                (B) The column is then washed with acetonitrile, which acts to both “push” the reagent (acid) onto the column, and to wash unreacted reagent and the removed 5′-blocking group (e.g. trityl alcohol) from the column.        (C) The primer support is then reacted with a phosphitylation reagent (amidite), which is dissolved in acetonitrile, the amidite having the formula:        
wherein bg is a 5′-blocking group, 1 g is a leaving group, G is O or S, pg is a phosphorus protecting group, and R2′ and Bx have, independent of the analogous variables on the primer support, the same definitions as previously defined.
The product of this reaction is the support-bound phosphite dimer:
Support-bound wherein each of the variables bg, pg, G, R2′ and Bx is independently defined above, link is the linker and supt is the support, as defined above.                (D) The support-bound dimer is then typically washed with acetonitrile.        (E) A capping reagent in acetonitrile is then added to the column, thereby capping unreacted nucleoside.        (F) The column is then washed again with acetonitrile.        (G) The support-bound dimer is then typically reacted with an oxidizing agent, such as a thiolating agent (e.g. phenylacetyl disulfide), in acetonitrile, to form a support-bound phosphate triester:        
                wherein G′ is O or S and the other variables are defined herein.        (H) The support-bound phosphate triester is then typically washed with acetonitrile.        
Steps (A)-(F) are then repeated, if necessary, a sufficient number of times to prepare a support-bound, blocked oligonucleotide having the formula:
wherein n is a positive integer (typically about 7 to about 79).
The phosphorus protecting groups pg are then typically removed from the oligomer to produce a support-bound oligomer having the formula:
which, after washing with a suitable wash solvent, such as acetonitrile, is typically cleaved from the solid support, purified, 5′-deblocked, and further processed to produce an oligomer of the formula:

The person having skill in the art will recognize that G′H bound to a P(V) phosphorus is generally is ionized at physiologic pH, and that therefore, wherever G′H appears in the formulae above, or hereafter, G′− is synonymous therewith (the O− or S− being countered by a suitable cation, such as Na+).
A typical blocking group for 5′-protection of nucleotides is the dimethoxytrityl group (DMT). The DMT group is acid labile, and may be removed with relatively weak acid, such as dichloroacetic acid. It is important that the oligonucleotide be produced in both good yield and excellent purity. Yield is commonly expressed in terms of coupling efficiency, which is a measure of the degree to which each successive monomer is coupled to the extant oligonucleotide. Coupling efficiency is affected by a number of factors, including the choice of nucleoside monomers, solvents, temperature, reagents, etc.
Purity is affected by a number of factors, including incomplete coupling (which produces so-called short-mers), as well as the introduction of impurities by reagents, solvents, etc.
It is a goal of oligonucleotide synthesis to produce large quantities of oligonucleotides in excellent yield and purity. Despite advances in the art of oligonucleotide synthesis, there is still a need for synthetic methods the produce oligonucleotides of improved purity.